This invention concerns an apparatus for determining the base sequence of a nucleic acid and, more specifically, it relates to a continuous electrophoresis detection system for nucleic acid fragments capable of determining the base sequence of a nucleic acid continuously at a high accuracy and higher speed.
The base sequence of a nucleic acid has heretofore been determined, for example, by Maxam-Gilbert's method (Methods in Engymology 65, pp. 495-701). In this method, a nucleic acid labelled with a radioisotope is chemically cut into fragments of the nucleic acid and then the fragments of different length are arranged in the order of their molecular weights in a gel support sandwiched between glass plates. Then, the gel support is detached from the glass plates for taking an autoradiogram therefor, by which the electrophoretic band containing radioactive fragments of the nucleic acid is detected to determine the base sequence of the nucleic acid. Explanation will be made for the conventional method of determining the base sequence of the nucleic acid fragments by the prior art referring to the drawing. FIG. 1 is a perspective view for the structure of a conventional electrophoresis apparatus for the nucleic acid fragments. As shown in the drawing, the electrophoresis apparatus comprises an electrophoretic gel 2 for nucleic acid fragments sandwiched between two sheets of glass plates 3, electrolyte cells 1 for immersing therein both ends of the electrophoretic gel 2 and a DC power source 10. In this apparatus, when a specimen of nucleic acid fragments labelled with a radioisotope, (for example .sup.32 P) is supplied to the slot 5 of the electrophoretic gel 2 and subjected to electrophoresis under a voltage Ev of about 50 V/cm per gel length, the nucleic acid fragments having an identical molecular weight move from the cathode to the anode while forming an electrophoretic band 4 respectively and move electrophoretically with a mobility substantially in an adverse proportion to the logarithm of their molecular weight. The base sequence of the nucleic acid is determined in the order of the molecular weights based on the electrophoretic pattern of the electrophoretic bands 4 of the nucleic acid fragments.
In the case of the Maxam-Gilbert's method, the nucleic acid fragments supplied to the slot 5 in FIG. 1 have a chain length of the bases in an average number of 1000 in view of the limitation from the restriction enzyme employed. The number of the bases for the nucleic acid fragments that can be analyzed in one electrophoretic process is from 400 to 500 and remaining nucleic acid fragments with longer chain length can not sufficiently be moved electrophoretically but remain near the slot 5 of the electrophoretic gel 2 shown in FIG. 1. In this case, although they can be caused to flow from the cathode end of the gel 2 by continuing the electrophoresis for a longer period of time, this is not practical since the separation of the electrophoretic band for the nucleic acid fragments becomes insufficient.
Furthermore, in the case of the Chain Terminator Sequencing Method (Methods in Engymology, 65, pp. 299-494), since the nucleic acid fragments for electrophoresis prepared upon determination of the nucleic acid base sequence are a mixture comprising ingredients with more than 7000 bases and ingredients with less than several hundreds bases, the components with more than 7000 bases are scarcely electrophoretized but remain near the slot 5 of the electrophoretic gel 2 also in this case. Accordingly, it has been difficult in either of the Maxam-Gilbert's Method and the Chain Terminator Sequencing Method to continuously supply various kinds of nucleic acid specimens and analyze the base sequence of the nucleic acids.